Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization

Background This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. Methods KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. Results KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. Conclusion The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.


Introduction
Klebsiella pneumoniae, a Gram-negative, non-motile, encapsulated bacterium, is a member of the Klebsiella genus within the Enterobacteriaceae family.It is responsible for various infections including pneumonia, urinary tract infections, and nosocomial infections.Respiratory tract infections caused by K. pneumoniae are widespread and severe, with a high mortality rate reaching 50% [1].The situation is further complicated by the prevalence of antibiotic-resistant strains, with approximately 80% of the K. pneumoniae isolates being resistant to antibiotics, making treatment challenging [2].The rise of multidrugresistant strains, particularly those resistant to carbapenems, has become a significant issue in both hospital and community settings due to the extensive use of antibiotics and efficient transmission [3].Additionally, K. pneumoniae is notable for its ability to form biofilms, which contributes to its significance as a major pathogen in healthcare-associated infections [4].Biofilm formation in K. pneumoniae infections is a crucial virulence factor, facilitating its persistence in clinical settings.Biofilms provide protection against the host immune response and antibiotics, promoting the acquisition of resistance traits and the development of multidrug resistance (MDR) phenotypes.The dense extracellular matrix of biofilms physically obstructs antibiotic penetration, while metabolic changes render bacteria less susceptible to antibiotics targeting actively dividing cells.Additionally, biofilm-associated gene expression upregulates efflux pumps and stress response mechanisms, further enhancing antibiotic resistance [4][5][6].The emergence and rapid spread of multidrug-resistant strains of K. pneumoniae have posed a significant threat to public health, highlighting the urgent need for innovative approaches to combat these infections [7,8].Bacteriophages, or phages, represent a promising alternative to conventional antibiotics due to their specific targeting of bacterial pathogens [1][2][3][4][5][6][7][8][9].These viruses have gained attention for their ability to specifically target and kill bacterial pathogens, while leaving beneficial bacteria and human cells unharmed [2,[7][8][9][10].Phages are naturally occurring entities that infect and replicate within bacteria.They possess a high degree of specificity, with different phages targeting specific strains or species of bacteria [3][4][5][6][7][8][9][10].This specificity is attributed to the recognition and binding of phage tail structures to specific receptors on the surface of bacterial cells.One of the key advantages of phages over traditional antibiotics is their ability to evolve alongside bacteria.As bacteria develop resistance mechanisms against antibiotics, phages can adapt and evolve to overcome these resistance mechanisms [4][5][6][7][8][9].This dynamic nature of phages allows them to maintain their effectiveness against evolving bacterial pathogens [11] Moreover, phages have a unique mode of action compared to antibiotics.Instead of directly killing bacteria, phages replicate within the bacterial host, leading to the lysis and destruction of the bacterial cell.This lytic activity not only kills the targeted bacteria but also helps prevent the development of bacterial resistance [12].Phages offer several other advantages as well.They have a broad range of host specificity, allowing them to target a wide variety of bacterial pathogens [13].Additionally, phages can penetrate biofilms, which are protective structures formed by bacteria that make them highly resistant to antibiotics [14,15].By effectively targeting biofilms, phages provide a potential solution to chronic and persistent infections [5].Another benefit of phages is their relative safety.They have been extensively studied and used in certain regions for decades, particularly in Eastern Europe, as a therapeutic option for bacterial infections.Phages are generally well-tolerated by the human body and have minimal impact on the normal microbiota [10][11][12].Phage therapy, the use of phages to treat bacterial infections, has shown promising results in both in vitro and in vivo studies.It has demonstrated efficacy against multidrug-resistant bacteria, including those that are resistant to conventional antibiotics.However, further research is needed to fully understand the potential of phage therapy and optimize its use in clinical settings.The aim of this study is to characterize the genome sequence of phage KZag1 and investigate its potential as a therapeutic agent against biofilms of K. pneumoniae.

Bacterial strains and growth conditions
Clinical specimens suspected to contain Klebsiella isolates were collected from the sputum of patients at Zagazig University Hospital, Sharqia, Egypt, following the acquisition of informed consent.The collected samples were promptly transported to the laboratory for further processing.To ensure the safety of laboratory personnel and prevent cross-contamination, the samples were handled in accordance with standard biosafety protocols.Laboratory personnel wore appropriate personal protective equipment (PPE), including gloves and laboratory coats, during sample processing.
Serial dilutions of the collected samples were performed to achieve a suitable bacterial load for isolation.Using a sterile loop or spreader, the diluted samples were streaked onto selective agar plates specifically designed for the isolation of Klebsiella, such as MacConkey agar or blood agar (Oxoid, United Kingdom) [16].These plates were then incubated at the optimal temperature for Klebsiella growth, typically 35-37°C, for a period of 24-48 h.Following incubation, the agar plates were examined for the presence of bacterial colonies.Colonies displaying characteristic Klebsiella morphology, such as a mucoid appearance, lactose fermentation, and non-hemolytic on blood agar plates, was selected.Using a sterile loop, these selected colonies were streaked onto fresh agar plates to obtain pure cultures, ensuring the isolation of individual Klebsiella strains [17].The isolated colonies were then incubated under appropriate conditions to facilitate further growth and identification.To confirm the identity of the isolated Klebsiella strains, additional tests were conducted.Specifically, DNA extraction was conducted using the QIAamp DNA Mini Kit supplied from QIAGEN (USA) with Catalogue no.51304.Subsequently, 16S rRNA sequencing was performed using the ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA), with Cat.No. 4336817, provided by Sigma Company, located in Giza, Egypt, for the accurate identification of the Klebsiella strains [18].The GenBank accession number link provided: https:// www.ncbi.nlm.nih.gov/ nucle otide/ OP942 216.1.

Enrichment and Isolation of K. pneumoniae bacteriophage
Bacteriophages were isolated from different sewage water samples obtained from Sharkia Governorate, Egypt, using the enrichment technique [21].Initially, 100 mL of sewage was filtered through a 0.45μm-filter membrane and combined with an equal volume of nutrient broth in 500 mL Erlenmeyer flasks.Subsequently, 5 mL of fresh K. pneumoniae culture (2.0 × 10 8 CFU mL −1 ) was added to each sewage sample.The flasks were incubated on a shaker (120 rpm) at 37 ºC for 24 h.After incubation, the mixture was centrifuged at 10,000 rpm for 20 min, and the resulting supernatant was filtered through a 0.45μm-filter membrane to detect the presence of phages using spot test and plaque assay methods with K. pneumoniae as the host.Phage titration was performed by tenfold serial dilution of the samples in saline solution and spotting them on lawns of K. pneumoniae, with the phage titer expressed in plaque forming units (PFU) per mL.

Purification of K. pneumoniae bacteriophage
Phages were propagated and purified from various single-plaque isolates following Kim et al. [21].The isolated phages underwent five successive single-plaque isolations until homogenous plaques were obtained.In each isolation, a single plaque was picked and incubated with 1 mL of nutrient broth containing an overnight culture of K. pneumoniae at 37 ºC with agitation.After incubation, the phage-host mixture was centrifuged at 10,000 rpm for 10 min, and the supernatants were filtered through a 0.45μm Millipore filter to eliminate any bacterial contamination.The purified phages were stored at 4ºC for further characterization.

Electron microscopy analysis of isolated bacteriophage
The morphology and structure of the isolated bacteriophage were analyzed using transmission electron microscopy (TEM), according to the method described by Abdel-Haliem and Askora [22].The phage suspension was prepared, containing a concentration of 10 8 (PFU mL −1 ).A small volume of the phage suspension was applied onto Carbon-coated formvar films on 200 mesh copper grids.Excess liquid was carefully removed from the grid using filter paper.A few drops of the Sodium phosphotungstate solution were added onto the grid, covering the phage particles.Excess Sodium phosphotungstate solution was gently removed from the grid using filter paper.Images of the phage particles were captured using a Hitachi H600A electron microscope at the Faculty of Agriculture, Mansoura University, Egypt.

The one-step growth
The one-step growth curve of the phage was determined following a standard protocol [16].Briefly, a culture of the host bacterium K. pneumoniae was grown to the logarithmic phase of growth.The bacterial culture was then infected with the phage at a multiplicity of infection (MOI) of 0.01, which means that for every bacterium, only one phage was added.After allowing the phage to adsorb to the bacterial cells for a specific duration 5 min, the mixture was diluted and plated onto agar plates to determine the number of viable phages (PFU) at time zero.This represented the initial phage count.Subsequently, samples were taken at regular intervals every 5 min for certain duration 2 h.Each sample was immediately diluted and plated onto agar plates to quantify the number of infective phages at each time point.This allowed for the construction of the growth curve.To calculate the burst size, the number of new phages released from each infected bacterium was determined by comparing the phage count at the peak of the growth curve with the initial phage count.The burst size represents the average number of phages released per infected bacterium.The experiment was performed in triplicate to ensure the reliability of the results, and the average values along with standard deviations were reported.

Host range
The host range of Kzag1 phage was determined experimentally using a collection of bacterial strains, including K. pneumoniae and related species, as potential hosts.Each bacterial strain was cultured to the logarithmic growth phase, and 100 µL of each culture was spread on separate agar plates.After drying, small drops of the phage suspension were added to the agar surface to allow phage attachment to the bacterial cells.The plates were then incubated at the optimal growth temperature for the bacterial strains at 37°C.After an appropriate incubation period 18-24 h, the plates were inspected for the presence of plaques, indicating successful phage infection and subsequent bacterial cell lysis.The absence of plaques indicated that the phage could not infect the specific bacterial strain.

The influence of temperature and pH on the stability of the phage
The influence of temperature and pH on the stability of the phage was examined.Thermal stability tests were performed following the protocol described by Mahmoud et al. [23].Phage samples with a known titer (10 6 -10 8 PFU mL −1 ) were exposed to different temperatures ranging from 30°C to 100°C for 10-min intervals in a water bath incubator.The infectivity of the phages was determined immediately after incubation using the double-layer agar plate method.Additionally, pH stability tests were conducted by inoculating a known phage suspension (10 6 -10 8 PFU mL −1 ) into LB liquid medium obtained from Thermo Fisher Scientific, United States with pH values ranging from 3.0 to 12.0, followed by overnight incubation at 4°C.The viability of the bacteriophages was assessed using the overlay method as described by Adams [21].

The impact of the Kzag1 bacteriophage on the proliferation of K. pneumoniae
The impact of Kzag1 phage on the growth of K. pneumoniae was investigated.A culture of K. pneumoniae was diluted to a final density of 2.0 × 10 8 CFU mL −1 and placed in a nutrient broth, then incubated at 37°C for 24 h.The phage suspension was also diluted to achieve a MOI of 0.1.Subsequently, 100 µL of various phage concentrations was added to the bacterial suspension [24], and the mixture was incubated under sterile conditions.The survival of K. pneumoniae was assessed at intervals of 5, 10, 15, 20, 25, 30, and 35 min using the plaque assay method.

The impact of individual Zag1 phage on biofilm formation
To assess the impact of Zag1 phage on biofilm formation by Klebsiella, the following experimental procedures were conducted according to Jamal et al. [25].A biofilm was developed by introducing 200 μL of bacterial culture (10 8 CFU mL −1 ) into each well of a 96-well flat-bottomed polystyrene microtiter plate, followed by incubation at 37°C for 24 h with gentle agitation at 120 rpm.After the biofilm formation period, any excess fluid was discarded, and the wells were washed twice with 0.9% NaCl to eliminate unattached planktonic cells.The plate was then allowed to dry at 37°C for one hour.The isolated phages were diluted in 0.9% NaCl and added to the respective wells containing their host bacteria.Different concentrations of the Zag1phage was used, including MOI = 0.1, MOI = 1, and MOI = 5.The control wells were set up using uninoculated normal saline, and they remained untreated throughout the experiment.Specifically, the uninoculated normal saline serves as the negative control, while the well inoculated with bacterial culture serves as the positive control.The plates were incubated at 37°C with constant shaking at 120 rpm for an additional day after the establishment of the biofilm.Excess fluid was removed from each well, and the biofilms were washed as previously described.The wells were left to dry for one hour at 37°C.To determine the total biomass of the biofilm, staining with 1% crystal violet was performed for 20 min.The plates were then washed with distilled water and air-dried.Next, 200 μl of 0.9% NaCl solution was added to each well, and the absorbance was measured at OD570 using an ELISA plate reader (Biotek Synergy HT Microplate Reader, USA.Triplicate measurements were taken for both control and test samples.

Phage DNA sequencing and subsequent bioinformatics analysis
The phage DNA sequencing and subsequent bioinformatics analysis were carried out as follows: Initially, phage DNA was extracted using the phenol-chloroform method, following the protocol by Sambrook and Russell [26].The purified phage DNA was then subjected to sequencing using the Illumina Miseq platform (Illumina, San Diego, CA, United States).The obtained sequencing data was assembled using SPAdeS v.3.13.0 [27] by Sanigen Inc., South Korea.To identify ORFs, a combination of Glimmer3 [28], GeneMarkS [29], and the RAST annotation server [30] was utilized.The annotated data were organized using Artemis [31].Furthermore, the tRNA sequence within the phage genome was analyzed using the tRNAscan-SE program.Predictions for the functions of the phage proteins were made using NCBI BLASTp and the InterProscan program [32].The annotated genome sequence of the KZag1 phage was deposited in the NCBI GenBank database under accession number OR502445.Phylogenetic analysis of the Zag1 phage was conducted by querying the Blast database and reconstructing a phylogenetic tree.Genomic sequences of relevant phages were retrieved from the GenBank database (https:// www.ncbi.nlm.nih.gov/ genba nk/) [32].These sequences underwent alignment using Clustal Omega to ensure precise alignment, considering sequence homology and structural similarities [33].Subsequently, the aligned sequences were used to construct phylogenetic tree employing the robust maximum likelihood (ML) methodology [34].Statistical analyses were conducted to evaluate the significance of the inferred phylogenetic relationships.

Statistical analysis
Each experiment was conducted in triplicate, and the average of the triplicate determinations was taken to represent the results.Statistical analyses were carried out using SPSS software package version 11.5 and Microsoft Excel 2010.The data were subjected to analysis of variance, and significant differences (p > 0.05) between means were determined according to Pallant [35].

Antibiotic susceptibility of K. pneumoniae K9 (OP942216)
Among the 95 bacterial isolates obtained from sputum samples, 15 isolates (15.7%) displayed characteristic Klebsiella morphology, including mucoid appearance, lactose fermentation, and non-hemolytic on blood agar plates.These isolates were further selected for antibiotic susceptibility testing.The susceptibility of the isolated K. pneumoniae strains to various antibiotics was evaluated using the disc diffusion method.The results, shown in (Table 1), indicated that the K. pneumoniae strains exhibited resistance to more than two of the tested antibiotics.Notably, the strains showed resistance to multiple antibiotics, including gentamicin, sulfamethoxazole, tetracycline, amoxicillin/clavulanate, nalidixic acid, and cefotaxime.Among the tested isolates, K. pneumoniae K9, isolated from a sputum sample, displayed the highest Multiple Antibiotic Resistance (MAR) index, with a value of 1.0, indicating significant resistance to multiple antibiotics.Molecular identification of this isolate (K9) was performed by extracting genomic DNA, amplifying rRNA using PCR, and sequencing the amplicon.The obtained sequence was deposited in the GenBank under accession number OP942216.

Bacteriophage isolation and morphological characterization (TEM)
Bacteriophages specific to K. pneumoniae K9 were isolated from an enrichment culture containing K. pneumoniae.Sewage water samples were collected from two large sewage plants in Sharkia Governorate, following the procedures outlined in the materials and methods section.The spot and plaque assay methods were utilized to detect the presence of phages.Among the observed plaques, a single plaque was selected for further analysis.This plaque was designated as Zag1, and subsequent purification and characterization steps were conducted.Transmission electron microscopy (TEM) was employed to examine the morphology of the isolated phage, KZag1.KZag1 exhibits a typical Myovirus structure; featuring a 75 ± 5 nm diameter icosahedral head and a short tail measuring 15 ± 5 nm.The results demonstrated that KZag1 belongs to the Myoviridae family.(Fig. 1).

Host range of K. pneumoniae KZag1 phage
The infectivity of Zag1 phage was assessed against different strains of K. pneumoniae and other bacterial strains (Table 2).KZag1 phage displayed robust lytic activity specifically targeting K. pneumoniae (Table 2).However, the remaining tested bacterial strains showed resistance to infection by the isolated phage, underscoring the highly specific nature of KZag1 towards K. pneumoniae.

One-step growth curve of KZag1 phage
The one-step growth curve analysis of KZag1 phage revealed a characteristic pattern of phage infection and replication.The latent period, which represents the time between phage adsorption and the initiation of replication, was determined to be 20 min.This was followed by a  rise period lasting 35 min, during which the phage population experienced exponential growth (Fig. 2).The entire cycle of infection, from adsorption to the release of new phage particles, was completed in approximately 50 min.The burst size, indicating the number of phage particles released per infected host cell, was calculated to be 83 for KZag1 phage.These findings provide valuable information about the kinetics and efficiency of phage replication and release within the host bacterial population.

Effect of temperature and pH on KZag1phage stability
The stability of Zag1 phage was assessed under different temperature and pH conditions.The results indicated that the infectivity of KZag1phage remained largely unaffected by temperature, particularly up to 60 °C (Fig. 3A).
The phage exhibited a survival rate ranging from 50 to 80% after exposure to temperatures of 70 °C for 10 min, suggesting its thermostability.However, at higher temperatures, KZag1 phage lost its infectivity and ability to lyse K. pneumoniae K9.Regarding pH stability, KZag1 phage demonstrated relatively stable behavior within the pH range of 6 to 8 (Fig. 3B).At pH levels of 11 or higher and pH levels of 4 or lower, Zag1 phage completely lost its infectivity.Notably, the phage exhibited greater  stability and infectivity at pH 7.These findings highlight the resistance of KZag1 phage to temperature variations within a certain range and its sensitivity to extreme pH conditions.Understanding the stability of KZag1 phage under different environmental conditions is crucial for its potential applications and efficacy in controlling K. pneumoniae infections.

In vitro assessment of KZag1 lytic activity against K. pneumoniae K9 biofilm
In vitro experiments were conducted to assess the lytic activity of KZag1 phage against a highly multidrugresistant strain of K. pneumoniae.When K. pneumoniae K9 was used as the host in combination with the isolated Zag1 phage, remarkably different inhibition patterns were observed.After a 24-h incubation period, complete inhibition of bacterial growth was observed, demonstrating the potent inhibitory activity of KZag1 phage against K. pneumoniae.The activity of Zag1 phage against K. pneumoniae K9 biofilm formation was investigated using varying MOI values (0.1, 1, and 10).The results showed a significant decrease in the biofilm biomass when treated with Zag1 phage compared to the control group (Fig. 4) among the tested MOI values; an MOI of 10 exhibited the highest inhibition of K. pneumoniae K9biofilm formation by the phage (Fig. 4).These findings indicate the strong potential of Zag1 phage in targeting and reducing biofilm formation by K. pneumoniae K9.This suggests its potential as an effective biocontrol agent to combat K. pneumoniae infections.Further studies are warranted to explore its applicability and efficacy in real-world settings.

Genomic features of KZag1 phage
The genome of the KZag1 phage, belonging to the Myoviridae family, was analyzed in this study.The phage genome had a size of approximately 157 kilobase pairs (kbp) and was found to contain 202 predicted ORFs (Table 3).Comparative analysis revealed a high degree of similarity between the KZag1 phage and Klebsiella virus 0507KN21.The genomic analysis of KZag1 revealed several notable features (Fig. 5).The total length of the genome was determined to be approximately 157 kbp, which is relatively large compared to other characterized phages.Within this genome, 202 ORFs were identified and annotated.These ORFs encode proteins with various putative functions, including those involved in phage replication, DNA packaging, structural proteins, and host interaction.A comparative analysis of the KZag1 genome with Klebsiella virus 0507KN21 revealed significant similarity, as depicted in (Fig. 6).These two phages exhibited a high degree of nucleotide sequence identity and displayed conserved genomic organization, suggesting a close evolutionary relationship and shared ancestry.Phylogenetic analysis further elucidated this connection by revealing a close relationship between KZag1 and other phages: Klebsiella phage T751, and Klebsiella phage cp18 (Fig. 6).The scale bar of 0.01 indicates minimal evolutionary divergence among these phages, implying a recent common ancestor.This observation is reinforced by the fast minimum evolution method employed in the pairwise alignments, which underscores the genetic similarity and shared evolutionary history among these phages.The high number of ORFs in the KZag1 genome indicates a complex genetic composition.These ORFs likely play crucial roles in the phage's lifecycle, including host recognition, replication, and assembly.The presence of specific genes associated with DNA modification, recombination, and mobile genetic elements suggests the potential for genetic diversity and adaptation within the phage population.Furthermore, the similarity to Klebsiella virus 0507KN21 suggests a shared host range and similar strategies for infecting and propagating within K. pneumoniae.This finding is significant as it indicates that KZag1 may possess similar infectivity and therapeutic potential against K. pneumoniae biofilms.The genomic characterization of KZag1 provides valuable insights into its genetic makeup and potential functions.The presence of numerous ORFs, similarity to Klebsiella virus 0507KN21, and the identification of specific genes involved in phage-host interactions highlight the phage's ability to infect and replicate within K. pneumoniae.Further investigation into the specific functions and mechanisms of these genes will deepen our understanding of phage-host dynamics and facilitate the development of phage-based therapies against K. pneumoniae infections.

Discussion
The present study focused on the analysis of a novel bacteriophage, KZag1, which specifically infects biofilms formed by K. pneumoniae.The research aimed to characterize the genome sequence and investigate the properties and potential applications of this phage.The findings contribute to our understanding of phage-host interactions and provide valuable insights for the development of phage-based therapies against K. pneumoniae biofilms.The first important aspect examined in this study was the sensitivity of K. pneumoniae strains to different antibiotics.The results revealed a high level of resistance to multiple antibiotics, indicating the presence of MDR strains.This observation aligns with previous reports highlighting the challenge of treating K. pneumoniae infections due to antibiotic resistance.The emergence of MDR strains poses a significant threat to public health, underscoring the urgent need for alternative treatment strategies [36].To address this issue, the researchers isolated and characterized KZag1, a bacteriophage specifically targeting K. pneumoniae biofilms.The phage was successfully isolated from an enrichment culture containing K. pneumoniae, and its lytic activity against the bacterium was confirmed through spot and plaque assays.The morphological characterization using transmission electron microscopy revealed that KZag1 belongs to the Myoviridae family, possessing icosahedral heads and contractile tails.This classification provides important insights into the phage's structural characteristics, which may influence its infectivity and interaction with the host bacterium [37].The host range analysis demonstrated that KZag1 exhibited strong lytic activity against K. pneumoniae strains but did not infect other tested bacterial strains, indicating a narrow host range.The specificity of bacteriophages towards certain bacterial hosts is a  well-documented phenomenon and is influenced by various factors such as surface receptors, bacterial cell wall structures, and immune evasion mechanisms.In the case of bacteriophage KZag1, its strong lytic activity against K. pneumoniae strains while not infecting other tested bacterial strains suggests a high degree of specificity towards K. pneumoniae.This specificity can be attributed to the presence of specific receptor sites on the surface of K. pneumoniae cells that are recognized by the phage's tail fibers or other structural proteins.The absence or structural differences of these receptor sites in other bacterial species may prevent the attachment and subsequent infection by the bacteriophage.Furthermore, the genomic makeup of bacteriophage KZag1 likely contains genes encoding proteins that specifically target and interact with components unique to K. pneumoniae, contribto its strong activity this bacterial species.While the exact mechanisms underlying the narrow host range of bacteriophage KZag1 may require further investigation, previous studies on phage-host interactions have demonstrated similar patterns of specificity, emphasizing the importance of understanding the molecular determinants driving bacteriophage infectivity.While a broad host range is desirable for therapeutic phages, narrow host specificity can still be advantageous in certain scenarios [38].By targeting K. pneumoniae specifically, KZag1 may offer a more precise and effective approach for treating K. pneumoniae biofilm-related infections without disrupting the beneficial microbial flora.The one-step growth curve analysis provided insights into the dynamics of phage infection.The latent period, generation time, and burst size of KZag1 were determined.The relatively short latent period of 20 min followed by a rise period of 35 min suggests that the phage has a rapid replication cycle.The burst size, which ranged from 83 to 100 phages per cell, indicates the potential for efficient phage-mediated lysis of K. pneumoniae biofilms.These findings provide valuable information for optimizing the therapeutic application of KZag1, such as determining the appropriate timing and dosage for effective treatment.Phage stability is a crucial factor to consider when developing phage-based therapies [39].The study investigated the effect of temperature and pH on the stability of KZag1.The results demonstrated that the phage remained infective and capable of lysing K. pneumoniae even after exposure to temperatures up to 60 °C, indicating its thermostability.Additionally, KZag1 exhibited optimal stability within a pH range of 6-8.However, extreme pH conditions, either highly acidic or alkaline, resulted in the loss of phage infectivity.These findings highlight the importance of considering environmental conditions when utilizing KZag1 as a therapeutic agent [39].The characterization of KZag1 and its demonstrated activity against K. pneumoniae K9 biofilms contribute to the growing body of knowledge on phagebased therapies.By specifically targeting biofilms, which are notoriously resistant to conventional antibiotics, KZag1 offers a potential alternative for combating K. pneumoniae infections.The narrow host range of KZag1 may limit its application to K. pneumoniae strains only, but it also reduces the risk of impacting the natural microbiota.Moreover, the phage's thermostability and stability within a physiological pH range enhance its potential for practical application.The analysis of the KZag1 phage genome, with a size of approximately 157 kilobase pairs (kbp) and belonging to the Myoviridae family, revealed several intriguing findings.The presence of 202 predicted ORFs within the genome suggests a complex genetic composition with various functional elements.The results of our phylogenetic analysis shed light on the evolutionary relationships between KZag1 and Klebsiella virus 0507KN21, along with other related Klebsiella phages: Klebsiella phage T751, and Klebsiella phage cp18, suggesting a close evolutionary connection and shared genetic ancestry [40].Such similarity in genomic organization and sequence conservation suggests common strategies for infecting and interacting with K. pneumoniae, the host bacterium.These findings offer valuable insights into the evolutionary dynamics of bacteriophages within the Klebsiella genus, crucial for understanding their evolutionary trajectories, host specificity, and potential applications in phage therapy and biotechnology.The abundance of ORFs in the KZag1 genome provides insight into the genetic diversity and complexity of the phage.These ORFs likely encode proteins involved in essential processes such as phage replication, assembly, DNA packaging, and host interaction.Detailed functional analysis of these ORFs can shed light on the mechanisms underlying the phage's lifecycle and its interaction with the host bacterium.Furthermore, the identification of specific genes associated with DNA modification, recombination, and mobile genetic elements within the KZag1 genome suggests potential mechanisms for genetic variation and adaptation.These elements may contribute to the phage's ability to evolve and overcome bacterial defense mechanisms, as well as facilitate the exchange of genetic material with other phages or bacterial hosts.The similarity to other Klebsiella phages, including Klebsiella virus 0507KN21, is particularly significant in the context of phage-based therapies [41].The close genetic relatedness between these phages suggests that they may share similar host ranges and infection mechanisms.This similarity provides promising prospects for the development of phage cocktails or combination therapies targeting K. pneumoniae infections, including those associated with biofilms.
Further studies are warranted to explore the functional significance of the identified ORFs and their role in phage-host interactions.Comparative genomics and proteomics approaches can provide insights into the shared and unique features of the related Klebsiella phages [42,43].Additionally, assessing the efficacy of these phages in biofilm eradication and infection control is crucial to evaluate their potential as therapeutic agents.In conclusion this study provides valuable insights into the characterization and potential therapeutic application of KZag1, a novel bacteriophage targeting biofilms formed by K. pneumoniae.The findings contribute to our understanding of phage-host interactions and highlight the potential of phage-based therapies as an alternative to combat antibiotic-resistant infections.Further research and clinical trials are warranted to evaluate the efficacy and safety of KZag1 and its potential integration into clinical practice for the treatment of K. pneumoniae biofilm-related infections.

Fig. 1
Fig. 1 Morphological Characteristics of K. pneumoniae Phage KZag1 (A) Phage Zag1 Plaque Morphology NA double-layer agar plate showing the plaque morphology of phage Zag1.Clear zones indicate areas where phage KZag1 has lysed the host K. pneumoniae cells.B Transmission Electron Microscopy (TEM) Image of Phage Zag1 shows three individual phage Zag1 particles, with a scale bar of 0.5 µm.C Transmission Electron Microscopy (TEM) Image of Phage Zag1 High-resolution TEM image revealing the detailed structure of phage Zag1.Scale represents 100 nm.D Phage Zag1 Adsorption on K. pneumoniae Cell Visual depiction of phage Zag1 attached to the surface of a K. pneumoniae bacterial cell

Fig. 2 Fig. 3
Fig. 2 Single-step growth curve for K. pneumoniae KZag1 phage.The plaque forming units (PFUs) per infected cell in cultures of K. pneumoniae K9 at different time post infection are shown.Samples were taken at intervals every 10 min

Fig. 4
Fig. 4 Phage Treatment of K. pneumoniae K9 Biofilm.The figure demonstrates the impact of Kzag1 phage treatment on K. pneumoniae bacterial biofilms using different multiplicities of infection (MOIs) of 0.1, 1 and10.Each data point on the graph represents the mean of three independent experiments.The results indicate the efficacy of phage treatment in reducing K. pneumoniae biofilm formation at varying MOI values

Fig. 5
Fig. 5 Whole genome map of phage Zag1.The figure displays the whole genome map of phage Zag1.The circular representation showcases the phage's genomic sequence, indicating the positions of various ORFs and functional elements.The map highlights key features and regions of interest within the phage's genetic structure

Table 1
Antibiotic Susceptibility using disc diffusion method for Klebsiella pneumoniae

Table 2
Host Range of K. pneumoniae KZag1 phage

Table 3
Annotation table CDS Position of KZag1